Cardiomyocyte differentiation in embryonic stem cells is poorly defined and a relatively nonselective process. hES cells and mouse embryonic stem (mES) cells have intrinsic differences in their response to biochemical factors (1). Consistent with these observations is the finding that cardiomyocyte differentiation between human ES cells or mouse ES cells or embryonic carcinoma (EC) cells is indeed dissimilar (2).
Differentiation of mouse embryonic stem cells into cardiomyocytes has historically been achieved through spontaneous differentiation in serum-containing medium or through treatment with compounds like DMSO, retinoic acid, Bone morphogenetic proteins, Fibroblast growth factors or the broad and non-specific de-methylating agent 5-aza-deoxycytidine (3,4). Compounds such as 5-aza-deoxycytidine have also been used to induce differentiation of cardiomyocytes from other stem cell types (adult bone marrow or fat tissue (5,6)). This indicates that different types of stem cells could potentially be used as a source for the generation of cardiomyocytes for applications such as cell therapy, tissue engineering, pharmacological and toxicological screening, or etc, provided effective processes and methods for the derivation of cardiomyocyte are available.
Current published methods for forming cardiomyocytes from hES cells rely on spontaneous differentiation in media containing animal serum (e.g. fetal bovine serum or calf serum), a media component that is largely undefined and subject to batch-to-batch variations. An enhancement of cardiomyocyte differentiation under these conditions can be achieved by treatment with 5-aza-2′-deoxycytidine (7). The differences between mouse and human ES cells is exemplified by the observation that the addition of DMSO or retinoic acid has no detectable effect on cardiomyogenesis when hES cells are treated although these compounds have shown an inductive effect on cardiomyocyte differentiation from mouse ES cells (7). Spherical structures from hES cells termed embryoid bodies (EBs) resulted in the formation of beating areas and thus cardiomyocytes when differentiation was performed in serum-containing medium (7,8). Overall, these methods of differentiation rely on the presence of serum in the medium as the primary compound of the cardiomyocyte differentiation protocol and this does not lend itself to being a clinically useful and reproducible system.
It has recently been demonstrated that hES cells when co-cultured in a serum free medium in contact with a visceral endoderm-like cell line END2 resulted in the differentiation of cardiomyocytes and the appearance of beating areas (9). The majority of these hES cell-derived cardiomyocytes have a fetal ventricular-like cardiomyocyte phenotype based on their electrophysiological parameters (9). Although the bioactivity responsible for the induction of hES-derived cardiomyocytes under these co-culture conditions is under investigation it is important to note that the differentiation does not involve serum in the differentiation medium and occurs in a more defined and therefore more reproducible serum free medium (10).
Using feeder-free conditioned media (CM) allows for a more clean and controlled differentiation of cardiomyocytes than co-culturing. However, the CM from END2-cells (END2-CM) contains proteins and other molecules released from the mouse END2 cells and as such hES-derived cardiomyocytes cultured from END2-CM would be considered a “xenoproduct” for clinical purposes. Therefore, identification of the “factor(s)” produced by the END2 cell is of paramount importance to the development of a hES-derived therapeutic product.
Identifying the END2 inducing factors and other factors involved in the differentiation process has been challenging and elusive since the generation of the visceral-endoderm-like END2 cell line from a mouse P19 embryonic carcinoma cell line. Earlier data has suggested that the END2 “factor” is secreted and it is a protein. Regardless of its uncertainty on whether it is protein, it is important to identify the factor(s) if a therapeutically acceptable product is to be developed. Identifying cardiomyocyte inducing factors will provide opportunities to develop clinically-compliant populations of HES-derived cardiomyocytes.
If differentiation conditions can be established with defined culturing conditions, and without the potential presence of animal pathogens, hES-derived cardiomyocytes may be produced safely which are suitable for cardiomyocyte transplantation in patients with heart disease.
Accordingly, the invention seeks to identify factors that are involved in the process of cardiomyocyte differentiation and provide a culture system that is suitable for the induction of stem cells to cardiomyocytes and cardiac progenitors.